The long-term objective of our research is to augment the efficacy of the cancer treatment photodynamic therapy (PDT). In PDT, utilizing a photosensitizer, visible light, and oxygen, reactive oxygen species are generated in a cellular target that can result in apoptotic cell death. PDT induces the de novo production of creamed, a sphingolipid that contributes importantly to apoptosis. The underlying mechanisms of de novo ceramide generation during PDT apoptosis remain obscure. This poses an important problem; because without the complete understanding of how ceramide generation occurs, it is highly improbable that ceramide can be manipulated to advance PDT cytotoxic potential. The objective of this application is to determine the regulation of ceramide production and the role of ceramide-metabolizing enzymes in apoptosis after PDT. The central hypothesis is: PDT-induced de novo ceramide production is controlled via oxidative stress dependent ceramide metabolizing enzymes and promotes apoptosis. Two specific aims will test the hypothesis; (1) identify the enzymes involved in regulating de novo synthesis of ceramide during PDT. Sphingomyelin synthase and glucosylceramide synthase will be characterized as potential PDT molecular targets. (2) Determine the influence of oxidative stress on de novo ceramide production and apoptosis after PDT. Specifically, the role of superoxide dismutase-dependent superoxide in these processes will be investigated. To address the aims, cells overexpressing sphingomyelin synthase, glucosylceramide synthase and superoxide dismutases, respectively, will be generated. In addition, glucosylceramide synthase-null mouse melanoma cells and embryonic fibroblasts isolated from Mn superoxide dismutase-knockout mice will be used. The impact of the proposed work is that it is expected to lead to strategies that will selectively block enzymes that utilize ceramide to potentiate cellular ceramide levels and ultimately promote PDT efficiency.